The cullin-RING ligases (CRLs) represent the largest super-family of multi-subunit E3 ubiquitin ligase complexes in eukaryotic cells. Organized in a modular form with a cullin-RING catalytic scaffold, an adaptor, and an interchangeable substrate-receptor subunit, the CRL1-5 complexes regulate a wide variety of cellular processes by promoting ubiquitination of a large number of protein substrates. Dysregulation and malfunction of CRLs are implicated in tumorigenesis, cancers, and a variety of other human diseases. This project focuses on structure-function studies of several CLR complexes in the regulation of cell growth, DNA replication, DNA repair, oxidative stress response, and hormone perception. In Aim1, a structural biology approach will be used to investigate how the substrate receptor subunits (DCAFs) of a newly identified family of CLRs, the CRL4 E3 complexes, are recruited to the ubiquitin ligase machinery. This study is aimed at resolving the current controversy over the identity of the CRL4 substrate receptors among all WD40-repeat proteins in the human genome. In Aim 2, a combination of biochemical and structural biology approaches will be employed to analyze the structural and functional roles of a novel functional ubiquitin E3 variant protein in the CRL4-Det1 E3 complex, which ubiquitinates the bZIP family of transcription factors such as c-Jun. In Aim 3, the crystal structure of the full length Keap1 dimer will be determined to elucidate how the BTB-domain protein functions as an oxidative stress sensor as well as a dimeric substrate receptor subunit of the CRL3 E3 complex. In Aim 4, the structural mechanism by which the plant COI1 F-box protein functions as the jasmonate plant hormone receptor will be investigated. Together with the recently published results of how another plant F-box protein TIR1 recognizes the plant hormone auxin, this study is aimed at establishing the structural principles of how these naturally occurring small molecules regulate the CRL E3s as agonists and laying the foundation of ongoing and future efforts of developing anti-cancer drugs targeting human CRLs.